1. Summary Mycobacterium tuberculosis isof deaths resulting from a single the leading cause infectious disease with 1.7 million victims annually. The exceptionally low permeability of the outer membrane contributes to the intrinsic resistance of mycobacteria to many antibiotics. Despite the well-documented importance of outer membrane proteins for nutrient uptake, secretion, and host-pathogen interactions in Gram-negative bacteria, only the porin MspA ofM. smegmatis the channel-forming protein OmpA of and M. tuberculosis have been characterized as mycobacterial integral outer membrane proteins. By contrast,E. coli more than 60 proteins to functionalize its outer uses membrane, none of which has significant sequence similarity to anyM. tuberculosisprotein. Rv1698 ofM. tuberculosiswas discovered by us as an outer membrane channel protein with unknown function. Intracellular copper in anM. tuberculosismutant lacking Rv1698 was 100-fold increased. AnM. smegmatis lacking the close homolog mutant _ re copper than the wild-type, while uptake o g Msm 3747 accumulated 11-fold mo f lucose remained unchanged. These results demonstrated that Rv1698-like channel proteins are required for copper efflux across the mycobacterial outer membrane and that secretion of Cu+ a mechanism by which isM. tuberculosis maintains copper homeostasis to prevent copper toxicity. Rv1698 is the first identified mycobacterial channel protein that is involved in efflux across the outer membrane. In addition, Rv1698 lacks a predicted copper binding motive and there is no energy source in the outer membrane that would support efflux trough the Rv1698 channels against the concentration gradient. Thus, Rv1698 is likely recruited by Cu+ specific inner membrane translocases that determine substrate specificity and provide energy for the transport. These findings indicate that mycobacteria possess multicomponent efflux systems that are functionally similar to those of Gram-negative bacteria. We also found thatM. tuberculosis not grow at did Cu2+ µM. The amount of copper in phagosomes of concentrations above 25 macrophages stimulated with interferon- increases to similar concentrations after infection withM. tuberculosis. Thus, macrophages appear to utilize copper to control intracellular growth ofM. tuberculosis. Uptake pathways for the essential micronutrient copper are unknown in mycobacteria. However, anM. smegmatis porin mutant did not grow with trace amounts of copper (<1 µM), but was more resistant than wild-type, demonstrating that channel proteins are required for copper uptake across the outer membrane. These outer membrane channels are essential components of a considerably revised model of copper homeostasis inM. tuberculosis. The implications of these findings for our understanding of transport mechanisms and, in particular efflux systems, in mycobacteria are profound.

Introduction

3

2. Introduction 2.1. The genus Mycobacterium 2.1.1. Taxonomy Mycobacteria are Gram-positive aerophilic bacteria with a high G+C content and show a rough morphology with uneven formed branched cells. Taxonomically, mycobacteria belong to the genusMycobacterium is the single genus within the family of which Mycobacteriaceaeorder Actinomycetales. This order includes various the in microorganisms, but mycobacteria and allied taxa are easily distinguished by their ability to synthesize mycolic acids (Rastogi et al., 2001). Mycobacteria possess the longest mycolic acids consisting of up to 90 carbon atoms (Barryet al.,1998) which confer acid-fastness to these bacilli. The genus is divided in slow- and fast-growing mycobacteria, which corresponds to phylogenetic data derived from 16S rRNA sequences (Rogall et al., Fast-growing species with generation times of less than 5 hours are mostly 1990). non-pathogenic, saprophytic soil bacteria such asMycobacterium smegmatis,M. phleiandM. chelonae. Slow-growing species have generation times of 20 hours and longer and are often pathogenic such asM. tuberculosis, the causative agent of tuberculosis (TB) andM. leprae, the pathogen causing leprosy. 2.1.2. Evolutionary pathway of the tubercle bacilli Speciation of recent members of theM. tuberculosis is estimated to have complex occurred during the last 15,000 to 20,000 years (Kapur et al., The complex 1994). consists ofM. tuberculosis,M. canettii,M. africanum,M. microtiandM. bovis(including M. bovisBCG). All members have identical rRNA sequences (Boddinghauset al.,1990; Broschet al.,2001) and exceptional little sequence variation resulting in 99.9% identity of their genomes at the nucleotide level (Sreevatsan al., et Musser 1997; al., et 2000). The subspecies can only be distinguished by a few phenotypic and genotypic characteristics but show great variety in terms of host range and pathogenicity (Broschet al., 2001; Brosch al., et 2002).Before genome sequences were available it wasbelieved thatM. tuberculosisevolved fromM. bovisby adaptation of an animal pathogen to the human host (Gonzalez-Flecha and Demple 1995). However, analysis of the genomes revealed thatM. bovis undergone numerous deletions relative to hasM. tuberculosisto be part of a separate lineage represented by therefore seems andM. africanum,M. microtiandM. bovis. This group is defined by successive loss of DNA in

Introduction 4 relation toM. tuberculosis in decreasing genome sizes (Garnier resulting et al., 2003). There are 14 regions of difference (RD1-14) that are absent inM. bovisBCG relative to M. tuberculosis 10 of those regions have been used as evolutionary markers to and propose the evolutionary pathway of the tubercle bacilli within theM. tuberculosiscomplex (Fig. 2.1) (Broschet al.,2002). 

Figure 2.1:Scheme of the proposed evolutionary pathway of the tubercle bacilli. The scheme is based on the presence or absence of conserved deleted regions and on sequence polymorphisms in five selected genes. Blue arrows indicate that strains are characterized bykatG463. (Leu), CTGgyrA95 ACC (Thr), typic9a5tecadiinsworraneerG.snismorgap1grouofrlA.ThThr) that strains belong to group 2 characterized bykatG463CGG (Arg),gyrA ( e CC red arrow indicates that strains belong to group 3, characterized bykatG463 (Arg), CGGgyrA95 (Ser), as defined by Sreevatsan AGC al., et 1997. The figure was taken from Broschet al.,2002. 2.1.3. Medical relevance of mycobacteria Mycobacteria are of great importance becauseM. tuberculosis the leading cause of is deaths resulting from a single infectious disease with 9.2 million new cases and 1.7 million deaths in 2006 (world health organization report 2008). The world health organization estimates that one third of the worlds population is infected and about 5 to 10% of infected people will become sick or infectious during their lifetime (WHO report, Factsheet No 104, revised 2007). Infection withM. tuberculosis not lead does

Introduction 5 unavoidably to disease, since the immune system can control the bacilli in check forcing them to adapt to prolonged periods of dormancy in tissues (Wayne 1994). It is suggested that the ability to shift down into non-replicating stages is crucial for the ability of tubercle bacilli to be dormant in the host for years or decades (Wayne and Hayes 1996). Immunocompetent individuals harboring latentM. tuberculosis carry % a 2-23 lifetime risk of reactivating tuberculosis, while patients with HIV reactivate tuberculosis at a much higher rate (Parrish et al., 1998). A problem is the rise of multi-drug resistant (MDR) strains (Bleed et al., 2001) which are resistant against at least rifampicin and isoniazid. Nearly 5% of all new infections are caused by MDR strains causing 500,000 tuberculosis cases per year. In addition, an extensively drug-resistance (XDR) form of tuberculosis has been reported in 45 countries. XDR tuberculosis is virtually untreatable and seriously threatens control efforts. Treatment of tuberculosis is difficult since only a few antibiotics are effective against fully susceptibleM. tuberculosis strains. Chemotherapy takes up to 6 month and must be extended for up to 2 years for MDR tuberculosis. The fact that many antibiotics do not affectM. tuberculosis can mostly be attributed to the unique mycobacterial cell wall which shields the mycobacterial cell from antibiotics and other toxic molecules due to its very low permeability (Brennan and Nikaido 1995). 2.2. The mycobacterial outer membrane and its proteins Due to its paramount importance as a pathogen, the growth and nutritional requirements ofM. tuberculosishave been intensively studied since its discovery more than a century ago (Koch 1882). However, nutrient transport inM. tuberculosisis still poorly understood despite a wealth of genomic data (Niederweis 2008). This is true in particular for transport processes across the outer membrane. The outer membrane ofM. bovisBCG and ofM. smegmatiswas visualized by cryo-electron microscopy (Fig. 2.2) and showed that mycolic acids are an essential component of this unusual supported lipid bilayer (Hoffmann et al., Mycolic acids are believed to form the inner leaflet of an 2008). asymmetrical bilayer while lipids that are extractable by organic solvents are assumed to form the outer leaflet (Minnikin 1982). X-ray diffraction of isolated mycobacterial cell walls showed that the mycolic acids are oriented parallel to each other and perpendicular to the plane of the cell envelope (Nikaido 1993). Its unique architecture and composition raise the question of how the mycobacterial outer membrane is

Introduction 6 functionalized for nutrient uptake, signal transduction, efflux of toxic compounds and secretion of material to the cell surface and to the extracellular medium.

Figure 2.2:Cryo-electron tomography ofM. bovisbacillus CalmetteGuérin (A,B,D),M. smegmatis(E), andE. coli(C). (A) Intact cell rapidly frozen (vitrified) in growth medium and imaged by using low-dose conditions at liquid nitrogen temperature. Black dots represent gold markers. (BE) Calculatedxy slices extracted from subvolumes of the three-dimensionally reconstructed cells and corresponding density profiles of the cell envelopes. The fitted Gaussian profiles in C (dashed curves) indicate the positions of the peptidoglycan (PG) and the outer membrane (OM). (D andE) Subtomograms recorded at nominal 6-μm defocus and reconstructed without noise reduction. CM, cytoplasmic membrane; L1 and L2, periplasmic layers; MOM, mycobacterial outer membrane. (Scale bars: A, 250 nm;BandC, 100 nm;DandE, 50 nm.) The picture was taken from Hoffmannet al.,2008. 2.2.1. Transport processes across mycobacterial outer membranes The mycobacterial cells are surrounded by an inner and an outer membrane (Hoffmannet al., Zuber 2008; et al., 2008) because of which it was proposed that nutrient uptake systems in mycobacteria are functionally analogous to those of Gram-negative bacteria. Three general pathways for transport processes across outer membranes exist (Fig. 2.3) (Niederweis 2008). (i) Hydrophobic compounds penetrate the membrane by temporarily dissolving in the lipid bilayer which is referred to as the lipid pathway. (ii) Polycationic compounds are believed to mediate their own uptake possibly by disorganizing the outer membrane locally. (iii) Small and hydrophilic compounds diffuse through channel proteins across the outer membrane. The outer membrane represents an extraordinary permeability barrier that protects mycobacteria from many toxic compounds and plays an essential role in virulence ofM. tuberculosis (Barry et al., 1998). Therefore, the contribution of porins to the permeability of the mycobacterial outer membrane is of great importance. It was shown previously that they promote the diffusion of nutrients like sugars (Stahl al., et 2001; Stephan al., et 2005) and inorganic phosphates (Wolschendorf al., etloss of porins generally contributes to 2007). In addition, the

Introduction 7 resistance against small and hydrophobic antibiotics as demonstrated for mycobacteria (Danilchanka al., et and numerous Gram-negative bacteria (Farra 2008) al., et 2008; Mammeriet al.,2008; Martinez-Martinez 2008; Rajaet al.,2008). It was proposed, that outer membrane proteins play a role in adaptation ofM. tuberculosisto its slower growth (DiGiuseppe-Champion and Cox 2007) and to its very different natural habitat.

2.2.2. Porin mediated diffusion of hydrophilic solutes inM. smegmatisThe discovery of MspA as the main porin ofM. smegmatis was proof of principle that porins do exist in mycobacteria despite the lack of proteins with sequence homology to known porins (Niederweis al., et The unique goblet-like structure of the single 1999). pore forming homooctameric MspA is in strong contrast to the homotrimeric organization of classical porins (Faller et al., such as OmpC where each subunit forms an 2004) independent channel by itself (Fig. 2.4). Its structural features define MspA as the first member of a new class of channel forming outer membrane proteins (Niederweis 2008). The chromosome ofM. smegmatisencodes four very similar porins designated MspA,

Introduction 8 B, C and D. The mature MspB, C and D proteins differ only at 2, 4 and 18 positions, respectively, from MspA (Stahlet al.,2001). Porin mutants ofM. smegmatisesseexprdan up to 75-fold lower permeability for glucose and grow much slower compared to wt demonstrating that porin-mediated influx of nutrients is a major determinant of the growth rate (Stephanet al.,2005). Besides its role in nutrient acquisition, the porin pathway is also utilized by many hydrophilic antibiotics such as ampicillin, fluoroquinolones and chloramphenicol (Stephan al., et Danilchanka 2004; al., et 2008) to cross the outer membrane ofM. smegmatis. Fig. 2.4:Structure comparison between the porins MspA (A, B) o M. smegmatisand OmpC (C, D) o E. coli. side view (A, C); top view (B, D). MspA consists of 8 identical subunits that form one central channel while each subunit o OmpC forms a channel.

2.2.3. The role ofM. tuberculosisOmpAin outer membrane permeability Porins are non-specific protein channels in bacterial outer membranes that enable the influx of hydrophilic solutes (Nikaido 2003). Channel-forming proteins have been found in cell wall extracts ofM. tuberculosis (Senaratne et al., Kartmann 1998; et al., 1999) andM. bovisBCG (Lichtingeret al.,1999) but these studies did not identify the proteins that actually account for the observed channels. However, a channel forming protein of M. tuberculosisto the OmpA protein family (Senaratnewas identified by its homology et al.,1998). OmpA-like proteins exist in all Gram-negative bacteria examined (Beheret al.,1980). In E. coli,OmpA is involved in conjugation (Schweizer and Henning 1977), in maintaining

Introduction 9 the structural integrity of the outer membrane (Sonntag al., et in resistance to 1978), environmental stresses (Wanget al.,2002) and in pathogenesis (Mittal and Prasadarao 2008). However, there is an ongoing debate about whether OmpA forms a pore.Although the channel forming ability of OmpA is well documented (Sugawara and Nikaido 1992; Aroraet al.,2000; Saintet al.,2000), crystaIs (Pautsch and Schulz 1998; Bond et al.,did not show an open water-filled channel. Furthermore, 2002)ompA mutantsofSalmonellaandE. colinot affect the outer membrane permeability (Bavoildid et al., Nikaido 1977; et al.,to many mutants lacking functional porins 1977) in contrast (Nikaidoet al.,1977; Harderet al.,1981; Sugawara and Nikaido 1994).Lipid bilayer experiments using OmpA ofM. tuberculosis expressed inE. colidemonstrated that the protein is able to form poresin vitro(Senaratneet al.,1998) butin vivo uptake experiments conducted with anompA mutant ofM. tuberculosis failed to conclusively demonstrate that OmpA is a major general porin ofM. tuberculosis(Solioz and Stoyanov 2003). The failure of theompAmutant to grow at low pH is likely the cause of the virulence defect in mice, because activated macrophages are able to override the block of phagosome acidification exerted byM.tuberculosisand to lower the pH inside phagocytic vacuoles (Schaibleet al.,1998). The strong induction ofompA transcription at low pH (30-fold) and in macrophages (5-fold) (Raynaud al., et 2002) suggests that acidification of the phagosome is the signal which triggers an OmpA-depending defense mechanism ofM. tuberculosisin macrophages to cope with growth-limiting proton concentrations. Thus, OmpA is one of the few protein virulence factors associated with the outer membrane ofM. tuberculosis(Solioz and Stoyanov 2003). 2.2.4. A proteome-wide screen for outer membrane proteins ofM. tuberculosisDespite the well-documented importance of outer membrane proteins for nutrient uptake, secretion processes and host-pathogen interactions in Gram-negative bacteria (Nikaido 2003), surprisingly few outer membrane proteins of mycobacteria are known. The only two well characterized examples of integral outer membrane proteins are the porin MspA ofM. smegmatis the channel-forming protein OmpA of andM. tuberculosis(Senaratneet al.,1998; Raynaudet al.,2002; Molleet al.,2006; Alahariet al.,2007). By contrast,E. coliuses more than 60 proteins to functionalize its outer membrane (Molloyet al.,which has significant sequence similarity to any none of 2000),M. tuberculosisprotein. The channel-forming protein MspA (Fig. 2.4 A, B) was shown to be located in the outer membrane ofM. smegmatis (Stahl et al., 2001) to provide classical porin